Principal Investigator/Program Director (Last, first, middle): Woodcock, Christopher, L. C.L.[unreadable]Woodcock[unreadable][unreadable][unreadable][unreadable][unreadable]MeCP2[unreadable]as[unreadable]a[unreadable]Chromatin[unreadable]Architectural[unreadable]Protein.[unreadable] [unreadable] PROJECT[unreadable]SUMMARY[unreadable] [unreadable] Mutations[unreadable]of[unreadable]the[unreadable]human[unreadable]MeCP2[unreadable]protein[unreadable]lead[unreadable]to[unreadable]the[unreadable]severe[unreadable]neurodevelopmental[unreadable]disorder[unreadable]Rett[unreadable] Syndrome[unreadable](RTT).[unreadable][unreadable]Misregulation[unreadable]of[unreadable]MeCP2[unreadable]is[unreadable]also[unreadable]seen[unreadable]in[unreadable]many[unreadable]of[unreadable]the[unreadable]Autism[unreadable]Spectrum[unreadable]Disorders.[unreadable][unreadable]MeCP2[unreadable]is[unreadable] a[unreadable]unique[unreadable]chromatin[unreadable]architectural[unreadable]protein[unreadable]that[unreadable]transmits[unreadable]the[unreadable]epigenetic[unreadable]information[unreadable]encoded[unreadable]in[unreadable]DNA[unreadable] methylation,[unreadable]and[unreadable]evidence[unreadable]suggests[unreadable]that[unreadable]in[unreadable]the[unreadable]context[unreadable]of[unreadable]chromatin[unreadable]binding[unreadable]it[unreadable]can[unreadable]cause[unreadable]local[unreadable]transcriptional[unreadable] repression[unreadable]both[unreadable]by[unreadable]its[unreadable]recruitment[unreadable]of[unreadable]histone[unreadable]deacetylase[unreadable](HDAC)[unreadable]complexes[unreadable]and[unreadable]by[unreadable]its[unreadable]innate[unreadable]ability[unreadable]to[unreadable] compact[unreadable]chromatin.[unreadable]This[unreadable]project[unreadable]is[unreadable]aimed[unreadable]at[unreadable]understanding[unreadable]these[unreadable]mechanisms[unreadable]and[unreadable]the[unreadable]root[unreadable]causes[unreadable]of[unreadable]the[unreadable] pathological[unreadable]effects[unreadable]that[unreadable]are[unreadable]seen[unreadable]with[unreadable]MeCP2[unreadable]mutations.[unreadable][unreadable]Recent[unreadable]work[unreadable]by[unreadable]the[unreadable]P.I.[unreadable]and[unreadable]collaborators[unreadable]has[unreadable]led[unreadable]to[unreadable] specific[unreadable]hypotheses[unreadable]regarding[unreadable]the[unreadable]domain[unreadable]organization[unreadable]of[unreadable]MeCP2,[unreadable]the[unreadable]functional[unreadable]associations[unreadable]between[unreadable] domains,[unreadable]its[unreadable]interactions[unreadable]with[unreadable]other[unreadable]proteins,[unreadable]and[unreadable]the[unreadable]molecular[unreadable]events[unreadable]leading[unreadable]to[unreadable]chromatin[unreadable]compaction.[unreadable][unreadable]This[unreadable] proposal[unreadable]describes[unreadable]experiments[unreadable]designed[unreadable]to[unreadable]test[unreadable]these[unreadable]hypotheses.[unreadable][unreadable][unreadable] In[unreadable]Aim[unreadable]1,[unreadable]spectroscopic[unreadable]techniques[unreadable]are[unreadable]proposed[unreadable]to[unreadable]compare[unreadable]the[unreadable]amount[unreadable]and[unreadable]type[unreadable]of[unreadable]secondary[unreadable] structure[unreadable]present[unreadable]in[unreadable]the[unreadable]wildtype[unreadable]domains[unreadable]of[unreadable]MeCP2[unreadable]with[unreadable]domains[unreadable]carrying[unreadable]RTT[unreadable]causing[unreadable]mutations.[unreadable][unreadable]Aim[unreadable]2[unreadable] employs[unreadable]similar[unreadable]strategies[unreadable]to[unreadable]complexes[unreadable]between[unreadable]MeCP2[unreadable]and[unreadable]DNA[unreadable]to[unreadable]test[unreadable]the[unreadable]hypothesis[unreadable]that[unreadable]DNA[unreadable]binding[unreadable] leads[unreadable]to[unreadable]changes[unreadable]in[unreadable]secondary[unreadable]structure.[unreadable][unreadable]Aim[unreadable]3[unreadable]is[unreadable]to[unreadable]test[unreadable]the[unreadable]hypothesis[unreadable]that[unreadable]phosphorylation[unreadable]of[unreadable]MeCP2[unreadable]results[unreadable] in[unreadable]weakening[unreadable]of[unreadable]MeCP2[unreadable]DNA[unreadable]interactions,[unreadable]and[unreadable]that[unreadable]this[unreadable]event[unreadable]plays[unreadable]a[unreadable]major[unreadable]role[unreadable]in[unreadable]its[unreadable]regulation[unreadable]in[unreadable]vivo.[unreadable][unreadable]This[unreadable] will[unreadable]be[unreadable]achieved[unreadable]by[unreadable]examining[unreadable]the[unreadable]interaction[unreadable]with[unreadable]DNA[unreadable]and[unreadable]chromatin[unreadable]of[unreadable]MeCP2[unreadable]mutations[unreadable]that[unreadable]mimic[unreadable] phosphorylation[unreadable]as[unreadable]well[unreadable]as[unreadable]MeCP2[unreadable]phosphorylated[unreadable]at[unreadable]selected[unreadable]sites.[unreadable][unreadable]Recent[unreadable]work[unreadable]shows[unreadable]that[unreadable]histone[unreadable]H1[unreadable]and[unreadable] MeCP2[unreadable]share[unreadable]overlapping[unreadable]binding[unreadable]sites[unreadable]on[unreadable]nucleosomes,[unreadable]suggesting[unreadable]that[unreadable]competition[unreadable]for[unreadable]binding[unreadable]sites[unreadable]may[unreadable] play[unreadable]an[unreadable]important[unreadable]regulatory[unreadable]role.[unreadable][unreadable]Aim[unreadable]4[unreadable]includes[unreadable]experiments[unreadable]to[unreadable]quantitate[unreadable]the[unreadable]competition[unreadable]between[unreadable]these[unreadable] two[unreadable]chromatin[unreadable]architectural[unreadable]proteins[unreadable]and[unreadable]examine[unreadable]the[unreadable]structural[unreadable]consequences[unreadable]on[unreadable]chromatin[unreadable]conformation[unreadable]of[unreadable] nucleosomal[unreadable]arrays[unreadable]which[unreadable]have[unreadable]both[unreadable]MeCP2[unreadable]and[unreadable]H1[unreadable]bound.[unreadable][unreadable]The[unreadable]co[unreadable]repressor[unreadable]protein[unreadable]mSin3A[unreadable]is[unreadable]a[unreadable]major[unreadable] binding[unreadable]partner[unreadable]of[unreadable]MeCP2,[unreadable]and[unreadable]also[unreadable]recruits[unreadable]HDACs.[unreadable][unreadable]However,[unreadable]little[unreadable]is[unreadable]known[unreadable]about[unreadable]the[unreadable]interactions[unreadable]with[unreadable] MeCP2[unreadable]and[unreadable]MeCP2[unreadable]mutants.[unreadable][unreadable]Aim[unreadable]5[unreadable]proposes[unreadable]an[unreadable]examination[unreadable]of[unreadable]these[unreadable]interactions,[unreadable]the[unreadable]structures[unreadable]of[unreadable]the[unreadable] protein[unreadable]complexes,[unreadable]and[unreadable]their[unreadable]impact[unreadable]on[unreadable]chromatin[unreadable]conformation.[unreadable][unreadable][unreadable] For[unreadable]common[unreadable]MeCP2[unreadable]mutations,[unreadable]there[unreadable]is[unreadable]a[unreadable]correlation[unreadable]between[unreadable]the[unreadable]severity[unreadable]of[unreadable]the[unreadable]disease,[unreadable]and[unreadable]the[unreadable] magnitude[unreadable]of[unreadable]effects[unreadable]seen[unreadable]with[unreadable]the[unreadable]assays[unreadable]used[unreadable]by[unreadable]the[unreadable]P.I.[unreadable][unreadable]This[unreadable]suggests[unreadable]that[unreadable]the[unreadable]proposed[unreadable]in[unreadable]vitro[unreadable]experiments[unreadable] will[unreadable]be[unreadable]relevant[unreadable]for[unreadable]understanding[unreadable]RTT.[unreadable][unreadable]Although[unreadable]this[unreadable]proposal[unreadable]is[unreadable]primarily[unreadable]aimed[unreadable]at[unreadable]understanding[unreadable]the[unreadable] defects[unreadable]in[unreadable]RTT[unreadable]causing[unreadable]mutations[unreadable]of[unreadable]MeCP2,[unreadable]it[unreadable]is[unreadable]hoped[unreadable]that[unreadable]the[unreadable]information[unreadable]we[unreadable]obtain[unreadable]will[unreadable]be[unreadable]used[unreadable]to[unreadable]select[unreadable] and[unreadable]screen[unreadable]for[unreadable]molecules[unreadable]that[unreadable]restore[unreadable]normal[unreadable]function,[unreadable]a[unreadable]strategy[unreadable]that[unreadable]is[unreadable]being[unreadable]successfully[unreadable]pursued[unreadable]with[unreadable]other[unreadable] DNA[unreadable]binding[unreadable]proteins.[unreadable][unreadable]The[unreadable]work[unreadable]will[unreadable]also[unreadable]contribute[unreadable]significantly[unreadable]to[unreadable]understanding[unreadable]a[unreadable]fundamental[unreadable]aspect[unreadable]of[unreadable] epigenetic[unreadable]control[unreadable]and[unreadable]chromatin[unreadable]remodeling.[unreadable][unreadable] Project Description Page 6